4.3. Flow Cytometry Gating Strategy

Figure 1A shows the gating strategy used to identify, count, and subtype circulating EVs. In detail, a forward scatter height (FSC-H)/ Side Scatter-H (SSC-H) dot-plot was used to establish a region, defined as “platelet free area”, containing the events with the EV scatter features (Figure 1A) [55,56]. Those events were represented on an LCD-H/Phalloidin-H dot-plot and EVs were identified as LCD positive/phalloidin negative dots (Figure 1B). Therefore, EVs (LCD+/Phalloidin- events) were analyzed on a CD45-H/CD41a-H dot-plot and CD45+ events were identified as leukocyte-derived EVs (Figure 1C) [18]. A CD45 negative logical gate was obtained, and the resulting population was represented on a CD31-H/CD41a-H dot-plot. Events showing the CD31+/CD41a+ phenotype were identified as platelet-derived EVs (Platelet EVs) (Figure 1D) [18], whereas the compartment was identified as endothelial-derived EVs (Figure 1D) [18]. Platelet-derived EVs (Figure 1E) and endothelial-derived EVs (Figure 1F) and all platelets (Figure 1G) were further analyzed for the activation marker CD62P. In Figure 1H, the used gating hierarchy is shown as a scheme.